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1 Copy number variation and microdeletions of the Y chromosome linked genes and loci across different categories of Indian infertile males Anju Kumari 1, Sandeep Kumar Yadav 1, M.M. Misro 2, Jamal Ahmad 3, Sher Ali 1* 1 Molecular Genetics Laboratory, National Institute of Immunology, New Delhi , India 2 Department of Reproductive Biomedicine, National Institute of Health and Family Welfare, New Delhi , India. 3 Centre for Diabetes and Endocrinology, JN Medical College and Hospital, Aligarh Muslim University, Aligarh, Uttar Pradesh , India Author affiliations Corresponding author Dr. Sher Ali alisher@nii.ac.in; sheralib5@hotmail.com Molecular Genetics Laboratory National Institute of Immunology New Delhi , India Ph

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6 sy1235 sy1260 sy1237 sy121 sy1322 sy280 sy1233 sy1682 sy627 sy142 sy1258 sy1161 sy1197 sy1191 sy1035 sy1318 sy254 sy1291 sy1125 sy1054 sy1190 sy1263 sy1206 sy1201 sy1246 Deletion recombination AZFc gr/gr b1/b2 b2/b3 P5-P1 proximal P5-P1 distal

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10 LEGENDS TO FIGURES Figure 1. Diagrammatic illustration showing analysis of the MSY region in the patients samples. (a) Represents human Y chromosome. HT indicate the heterochromatin region of Y chromosome; CEN, centromere and PAR, psuedoautosomal regions. (b) Regions on the Y chromosome where STS were analyzed. Sample IDs are given on the left. NFM represent normal fertile males; OS, oligospermic; AZ, Azoospermic and INS, Infertile males with normal spermiogram. Black solid lines relates to the samples indicating the presence of the STS analyzed, whereas the dotted lines represent deletion of the same.

11 Figure 2. STS mapping of AZFa region of the patients. STS mapping of AZFa region was done using samples from infertile males of different categories (oligospermic OS, azoospermic AZ, infertile males with normal spermiogram INS) and normal fertile males (NFM). Presence of the corresponding STSs in the patients is indicated by solid line and absence by dotted lines; patient IDs and their categories are shown on the left. As expected, all the STSs were found to be intact in the fertile male samples used as control. Figure 3. STS mapping of AZFb and AZFc regions. (a) Diagrammatic illustration of human Y chromosome. (b) Representative gels (i-viii) showing STS mapping of AZFb region for some patients. β-actin was used as an internal control. The STSs analyzed are shown on the right side and the patients IDs, in the bottom. NTC and PTC indicate the negative and positive controls, respectively. NFM, OS, AZ and INS indicate normal fertile males, oligospermic, azoospermic and infertile males with normal spermiogram, respectively. (c) AZFc STS mapping results of some of the representative samples from each category of males. Presence of the corresponding STSs (given on the top) in the patients are indicated by solid line and absence by dotted ones. The patient IDs are shown on the left side. Figure 4. Deletion frequency in the AZF regions in the infertile males from different categories. X-axis depicts the deletions in different regions of the Y chromosome, whereas the Y-axis shows % of the patients. Black bars indicate the % of males (belonging to particular category) with deletions and grey bars correspond to % of males without deletions. Panels a, b and c are for the OS, AZ and INS respectively. No deletions were observed in the normal fertile males. Figure 5. Copy number analysis of DYZ1 arrays by qpcr. Representative amplification plot (a) and standard curve (b) used for the copy number calculation of DYZ1 arrays. The bar graphs (c-f) show the distribution of copies in the males of different categories including oligospermic (43), infertile males with normal spermiogram (40), azoospermic (34) and normal fertile males (55) respectively. All the reactions were performed in triplicates and the results shown here are average of these triplicates.

12 Figure 6. Localization of DYZ1 on metaphase chromosomes, interphase nuclei and spermatozoa using FISH. (a) DYZ1 probe was labeled with Texas red, whereas metaphases and interphase nuclei were stained with DAPI. (b) Sperm nuclei are stained with DAPI. The Y bearing sperms are showing red signal of DYZ1, whereas the X bearing sperms lack the DYZ1 signal. (c) Male blood metaphase and (e) sperm samples were processed together with the experimental samples under identical conditions but not hybridized with the DYZ1 probe to exclude the background signal. (d) Female sample hybridized with DYZ1 probe. Patient IDs are shown in green. OS indicates oligospermic; AZ, azoospermic; INS, infertile males with normal spermiogram and NFM, normal fertile males. Figure 7. Copy number estimation of SRY, BPY2 and DAZ genes using real time PCR. (a-d) Summarizes the distribution of copy number of SRY, BPY2 and DAZ in oligospermic (a), Azoospermic (b), infertile males with normal spermiogram (c) and normal fertile males (d), respectively. X-axis depicts the gene of interest analyzed and the copy number assessed using real time PCR while the Y- axis indicates the % of males with corresponding copies for the genes. Grey bars indicate the normal copy number for the particular gene and the white bars indicate the copy number variation observed. A total of 43 OS, 34 AZ and 40 INS patients were analyzed for copy numbers and each reaction was set in triplicates during real time PCR amplification. Figure 8. Chromosomal localization of SRY gene on metaphases, interphase nuclei and spermatozoa using Fluorescence in situ hybridization (FISH). (a) The metaphase and interphase nuclei are stained with DAPI. The SRY gene localized on the Y chromosome is showing red signal and X-centromere, fluorescent green signal. (b) Figure represents mapping of SRY gene on the individual sperm. All the Y bearing sperms are shown in red, whereas those of X-bearing ones are shown in green. (c) Male blood metaphase and (e) sperm samples were processed under identical conditions but not hybridized with the SRY probe to exclude the background signal. (d) Female sample hybridized with SRY probe. The spermatozoa are stained with DAPI. Patient IDs are shown in yellow. AZ indicates azoospermic; OS, oligospermic; INS, infertile males with normal spermiogram and NFM, normal fertile male.